1. Field of the Invention
This invention relates to polynucleotides encoding viral polypeptides associated with the poultry disease known as Newcastle Disease and their preparation by recombinant DNA methods.
2. Description of the Prior Art
Newcastle disease virus (NDV) is a typical paramyxovirus and causes a severe respiratory infection in poultry. This disease is of great economic importance, requiring control by vaccination or quarantine with slaughter of all birds in confirmed outbreaks. The genome of NDV is a single strand of RNA of negative polarity, molecular weight 5.2-5.6.times.10.sup.6, or approximately 15,000 bases which is transcribed by a polymerase to produce viral mRNA. The genomic RNA is bound to three proteins in the viral nucleocapsid. These proteins are the nucleocapsid protein NP, the phosphoprotein P, and the large protein L. The nucleocapsid is contained within a lipid envelope derived from host cell plasma membranes on the inner surface of which is a shell of membrane or matrix protein M. On the outer surface of the viral envelope are the two viral glycoproteins, the haemagglutinin-neuraminidase HN, involved in the binding of the virus to host cells and the fusion protein F, involved in fusion with and penetration through the membrane.
The primary translation product of the F gene contains a signal sequence which is presumably removed during or shortly after translation of the polypeptide. Glycosylation of the polypeptide occurs as the polypeptide is translated to result in the precursor glycoprotein F.sub.o. The glycoprotein F.sub.o is usually cleaved during processing in vivo to give sub-unit polypeptides F.sub.2, F.sub.1, which are linked by a disulphide bridge. That is the sub-units F.sub.1 and F.sub.2 together form a single molecule because they are linked between cysteine residues by the --S--S-bond. The peptide bond cleavage correlates with virulence: F.sub.o of virulent strains is cleaved to F.sub.1 and F.sub.2 in a wide range of host cells, whereas F.sub.o of avirulent strains, such as LaSota and B1, is cleaved only in a restricted range of host cells. Except where F.sub.o is specifically referred to as such, or the context otherwise requires, the term F used herein includes both F and F.sub.o.
The primary translation product of the HN gene is the glycoprotein HN. In two avirulent strains a precursor glycoprotein (HN.sub.o) is cleaved to active HN by cleavage of a C-terminal glycopolypeptide. Except where HN.sub.o is specifically referred to as such, or the context otherwise requires, the term HN used herein includes both HN and HN.sub.o.
Monoclonal antibodies raised against the NDV HN glycoprotein neutralise NDV infectivity, R. M. Iorio and M. A. Bratt, J. Virology 48, 440-450 (1983). Y. Umino et al., Archives of Virology 81, 53-65 (1984) reported that monospecific antisera to the NDV HN glycoprotein were highly neutralising of haemagglutinin and neuraminidase activity. Antisera to the F glycoprotein inhibited haemolysis and virus-induced cell fusion and the combination of anti-HN and anti-F antisera appeared particularly effective in a plaque reduction assay. Accordingly it would be of interest to prepare, by recombinant DNA methods, artificial DNA or RNA encoding the NDV glycoproteins HN and F. (It is convenient herein to refer to DNA or RNA encoding the NDV proteins, although, strictly, it can encode only the polypeptide precursors thereof which are processed in vivo to glycoproteins). The primary products of NDV transcription in vivo are polyadenylated, capped and methylated mRNAs which are complementary to the genomic RNA and have sedimentation coefficients of 35S, 22S and 18S. The 18S and 35S transcripts code for the six proteins, the 18S RNA coding for proteins NP, P, M, HN, and F and the 35S RNA for the large protein L. The 18S RNA contains five distinct monocistronic poly A-mRNAs, i.e. each codes for one of the five proteins. S. R. Weiss et al, Journal of Virology 18, 316-323 (1976) have shown that these 18S mRNAs have relative molecular masses of 5.0, 5.7, 7.1, 7.4 and 8.5.times.10.sup.5.
P. L. Collins et al., Journal of Virology 43, 1024-1031 (1982) have performed in vitro translations and thereby have made assignments of the protein coded for by each mRNA. In ascending order of relative molecular mass they are M, P and proteins of r.m.m. 36K and 33K, NP, unglycosylated F and unglycosylated HN.
No definitive genomic map of NDV exists at present. Maps have been constructed by UV transcriptional mapping, as described by P. L. Collins et al., J. Virology 35, 682-693 (1980) and J. Virology 28, 324-336 (1978). These maps are inconsistent. The last published such paper indicated that the NP gene lies nearest the 3' end, followed by the P gene then the M and F genes in unknown order, followed by the HN gene, all in the 3' half of the genome, and the large L gene nearest the 5' end. However, UV transcriptional mapping is an imprecise technique which is known from work on Sendai virus to have given incorrect results.
Recently, L. E. Dickens et al., J. Virology 52, 364-369 (1984) have compared the gene order in human respiratory syncytial virus (a paramxyovirus) with the rhabdovirus VSV and NDV. The gene order disclosed (3' to 5') is NP, P, M, F, HN and L but it is derived from an unpublished personal communication from another scientist.
The SV5 genes encoding the HN and F proteins have been sequenced. See S. W. Hiebert et al., J. Virology 54, 1-6 (1985) and R. G. Paterson et al., Proc. Nat. Acad. Sci. USA 81, 6706-6710 (1984).
C. D. Richardson et al., Virology 105, 205-222 (1980), sequenced the first 20 amino acids at the N-terminal end of the F.sub.1 protein of Sendai Virus, SV5 and NDV. A mixture of all 18-mer oligonucleotides encoding the six consecutive amino acids of this NDV sequence with the lowest number of possible codons would have to contain a minimum of 864 different oligomers.
Recently, A. Wilde et al., Journal of Virology 51, 71-76 (1984) have shown that the 22S NDV RNA transcript gives rise to polycistronic RNA molecules. Their paper reports at page 75, R.H. column, lines 16-19 that cDNA clones derived from individual NDV genes were used to show that the RNA was bi- or tri-cistronic. No further information about such gene clones was given.
The Sendai virus HN and F genes have been sequenced by B. M. Blumberg et al., Journal of Virology 66, 317-331 (1985) and Cell 41, 269-278 (1985). Also Y. Hidaka et al., Nucleic Acids Research 12, 7965-7973 (1984) have sequenced part of the F gene.
3. The inventors' own prior disclosures
The present inventors have been engaged in cloning NDV genes by recombinant DNA technology. On Jan. 10th, 1985 at a Society for General Microbiology workshop at Birmingham, England, they presented orally an outline of their work including a short region of cDNA sequence thought to be in the region of the junction between HN and L genes. An open reading frame was not given.
On Jul. 17th, 1985, they gave an oral paper, with overhead projection, to a meeting of the Biochemical Society at Oxford, England. A brief abstract outlining the work but giving no detail, was circulated to delegates in about June 1985. A poster was exhibited in July 17th at the meeting. The poster reported the cloning in the plasmid pBR322 of a series of overlapping small fragments which by laborious and careful mapping techniques had been shown to span the entirety of the F and HN genes. The poster gave no nucleotide sequence information in the coding region but reported a deduced 69-residue amino acid sequence NPTSAVFD . . . PLLVEILKN near the 5' end of the HN gene. This sequence did not include any methionine or tryptophan residues which have unique DNA codons. The oral paper went no further in content than to the poster.
Despite the above reports, it was still impossible to specify with confidence the location on the NDV genome of the HN and F genes particularly in view of the unreliability of UV transcriptional mapping. Nor was there published any nucleotide sequence information which would enable the man skilled in the art to construct a small number of probes whereby the relevant genes in an NDV gene library could be hybridised to the probe and thence extracted from the library. Further, no such NDV gene library was known at the priority date of this patent application. Accordingly it remained a problem to prepare a cDNA or RNA coding for the HN and F genes of NDV.